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| United States Patent |
5,179,012
|
|
Gudin
, et al.
|
January 12, 1993
|
Process for the production and extraction of antioxidants from a
micro-organism culture
Abstract
The process consists of culturing in a closed photobioreactor (2)
microalgae suspended in a culture medium (1), the oxygen produced by the
microalgae by photosynthesis being collected and then reinjected into the
culture medium, separating (6) the microalgae from the culture medium (1),
dissolving (12) the microalgae, grinding or crushing (14) the solution of
microalgae, adding (16) a solvent to the ground or crushed solution for
solubilizing the antioxidants produced by the microalgae and then
separating (18) the liquid phases present.
| Inventors:
|
Gudin; Claude (Aix en Provence, FR);
Thepenier; Catherine (Manosque, FR)
|
| Assignee:
|
Commissariat A L'Energie Atomique (Paris, FR)
|
| Appl. No.:
|
638394 |
| Filed:
|
January 7, 1991 |
Foreign Application Priority Data
| Current U.S. Class: |
435/125; 435/41; 435/126; 435/136; 435/137; 435/170; 435/189; 435/822; 435/946 |
| Intern'l Class: |
C12P 017/06; C12P 017/04; C12P 009/02; C12R 001/89 |
| Field of Search: |
435/41,125,126,170,173,189,822,946,136,137
|
References Cited
U.S. Patent Documents
| 3997402 | Dec., 1976 | Michelson | 435/189.
|
| 4022604 | May., 1977 | Kawamura et al. | 435/125.
|
| 4029819 | Jun., 1977 | Michelson | 435/189.
|
| 4217728 | Aug., 1980 | Shimamatsu et al. | 47/1.
|
| 4320050 | Mar., 1982 | Rebeller et al. | 435/946.
|
| 4563349 | Jan., 1986 | Miyata et al. | 435/189.
|
| 4693842 | Sep., 1987 | Shilo et al. | 435/84.
|
| 4774185 | Sep., 1988 | Asami et al. | 435/822.
|
| 4906746 | Mar., 1990 | Barnier et al. | 435/72.
|
| 4978617 | Dec., 1990 | Furuya | 435/125.
|
| Foreign Patent Documents |
| A310522 | Apr., 1989 | EP.
| |
Other References
Chemical Abstracts, vol. 87, 1977 (Mistral et al.) No. 147729z.
Chemical Abstracts, vol. 74, 1971 (Antia et al.) No. 29131g.
|
Primary Examiner: Lilling; Herbert J.
Attorney, Agent or Firm: Pearne, Gordon, McCoy & Granger
Claims
We claim:
1. Process for the production and extraction of antioxidants, comprising
the following stages:
(a) culturing in a closed photobioreactor of photosynthetic microorganisms
suspended in a liquid culture medium for producing oxygen and
antioxidants, the oxygen produced by the microorganisms by photosynthesis
being collected and reinjected into the culture medium, said
microorganisms being chosen from the group consisting of rhodophyceae and
chlorophyceae, said antioxidants being chosen from the group consisting of
superoxide dismutase, vitamin C and tocopherols,
(b) separating the microorganisms from the culture medium,
(c) dispersing in a solution the microorganisms separated in (b),
(d) crushing the dispersed microorganisms,
(e) adding solvent to the solution obtained in (d) in order to solubilize
the antioxidants produced by the microorganisms and then
(f) separating the liquid and solid phases present.
2. Process according to claim 1, characterized in that stage (b) is
performed during an exponential growth phase of the microorganisms.
3. Process according to claim 1, characterized in that the microorganisms
are cultured under natural lighting conditions and in that stage (b) is
performed in the afternoon.
4. Process according to claim 1, characterized in that it comprises a stage
(1) of treating the culture medium (1) in order to increase the production
of antioxidants.
5. Process according to claim 1, characterized in that the solvent is an
organic solvent or vegetable or mineral oil.
6. Process according to claim 1, characterized in that stages (d), (a) and
(f) are performed at approximately 4.degree. C.
7. Process according to claim 1, characterized in that the liquid phases
obtained in (f) are concentrated and then purified.
8. Process according to claim 1, characterized in that the culture medium
separated in (b) is concentrated and then purified.
9. Process according to claim 1, characterized in that the solution of
stage (c) contains 20 to 100 g/l of dry microorganisms.
10. Process according to claim 1, characterized in that the oxygen produced
by photosynthesis is reinjected under pressure into the culture medium.
11. Process according to claim 1, characterized in that the microorganisms
are chosen from among Porphyridium cruentum and Haematococcus pluvialis.
12. A process for the production and extraction of antioxidants, comprising
the following steps:
(a) culturing in a closed photobioreactor of photosynthetic microorganisms
suspended in a liquid culture medium for producing oxygen and
antioxidants, collecting the oxygen produced by the microorganisms by
photosynthesis and reinjecting the collected oxygen into the culture
medium, heating said culture medium to increase production of
antioxidants, said microorganisms being chosen form the group consisting
of rhodophyceae and chlorophyceae, said antioxidants being chosen from the
group consisting of superoxide dismutase, vitamin C and tocopherols,
(b) separating the microorganisms from the culture medium,
(c) dispersing in a solution the microorganisms separated in step (b),
(d) crushing the dispersed microorganisms,
(e) adding solvent to the solution obtained in (d) in order to solubilize
the antioxidants produced by the microorganisms and then
(f) separating the liquid and solid phases present.
13. A process as claimed in claim 12, wherein said microorganism is chosen
from the group consisting of Porphyridium cruentum and Haematococcus
pluvialis and said solvent is an organic solvent.
14. A process as claimed in claim 12, wherein said heating step to increase
production of antioxidants is followed by subjecting the culture media to
ionization to further increase production of antioxidants.
15. A process as claimed in claim 4, wherein said microorganism is chosen
from the group consisting of Porphyridium cruentum and Haematococcus
pluvialis, said solvent is an organic solvent, said treating step is
heating the culture medium, and said antioxidants are superoxide-dismutase
enzymes.
16. A process for the production and extraction or antioxidants, comprising
the following steps:
(a) culturing in a closed photobioreactor of photosynthetic microorganisms
suspended in a liquid culture medium for producing oxygen and
antioxidants, collecting the oxygen produced by the microorganisms by
photosynthesis and reinjecting the collected oxygen into the culture
medium, subjecting said culture medium ionization to increase production
of antioxidants, said microorganisms being chosen from the group
consisting of rhodophyceae and chlorophyceae, said antioxidants being
chosen from the group consisting of superoxide dismutase, vitamin C and
tocopherols,
(b) separating the microorganisms from the culture medium,
(c) dispersing in a solution the microorganisms separated in step (b),
(d) crushing the dispersed microorganisms,
(e) adding solvent to the solution obtained in (d) in order to solubilize
the antioxidants produced by the microorganisms, and then
(f) separating the liquid and solid phases present.
17. A process as claimed in claim 16, wherein said microorganism is chosen
from the group consisting of Porphyridium cruentum and Haematococcus
pluvialis and said solvent is an organic solvent.
Description
The present invention relates to a process for the production and
extraction of antioxidants from a culture of photosynthetic microorganisms
suspended in a liquid medium, as well as the photobioreactor for culturing
these microorganisms. This process permits an intensive, controlled
production of antioxidants on an industrial scale.
The invention is applicable to the production of any antioxidant and in
particular to the production of superoxide-dismutase enzymes, known by the
abbreviation SOD of vitamin C, alpha, beta or gamma tocopherol, alpha
tocopherol being vitamin E.
Superoxide-dismutases are metal enzymes having two peptide subunits linked
to one another. There are three types of SOD classified as a function of
the nature of the metal:
Cu/Zn SOD, which are frequent in eukaryocytes--the traditional source of
said SOD being cattle blood and extraction takes place from erythrocytes;
Mn SOD found in eukaryocytes relative to mitochondria and in the
prokaryocytes;
finally, the Fe SOD which would appear to be specific to prokaryocytes i.e.
aerobic bacteria.
SOD's are dismutation catalysts of superoxide ions in accordance with the
reaction:
O.sub.2.sup.- +O.sub.2.sup.- +2H.sup.+ .fwdarw.H.sub.2 O.sub.2 +O.sub.2
Thus, SOD's are used in acute phenomena freeing superoxide radicals or in
aging phenomena. They are in particular used in the pharmaceutical fields
as anti-inflammatory agents, (rheumatism, arthritis), cosmetological
agents for protecting the skin and hair, e.g. for protecting against
ultraviolet rays, in the agro-alimentary field (for protecting oils and
fatty substances, as a regulating agent of the coloring or as fertilizers,
as well as in the biomedical field. Their use in cosmetology is in
particular described in FR-A-2 297 899.
The invention is applicable to any photosynthetic microorganism of the
microalgae type and the cyanobacteria type. One of the best known
cyanobacteria is the "blue alga".
Photosynthesis is the tranformation by solar energy of carbon dioxide into
primary hydrocarbon material, the oxygen being the main biproduct of this
biochemical transformation. This reaction can be symbolized by the
following Myers equation:
6.14 CO.sub.2 +3.65 H.sub.2 O+NH.sub.3 .fwdarw.C.sub.6.14 H.sub.10.3
O.sub.2.24 N+6.85 O.sub.2
The term C.sub.6.14 H.sub.10.3 O.sub.2.24 N corresponds to the
microorganisms, also known as biomass.
In FR-A-2 621 323 filed on Oct. 2, 1987 in the name of the Applicant is
described an apparatus for the intense, controlled production of
photosynthetic microorganisms. Said apparatus is in particular a closed or
sealed photobioreactor designed so as to control the monoculture of a
desired species, as well as the parameters of the culture, such as the
temperature, the pH, the CO.sub.2 and O.sub.2 pressures in the culture
medium, as well as the composition of the nutrient medium, in order to
favor the production of the selected species.
These photobioreactors essentially comprise a solar sensor or receiver
which is transparent to light in which flows the liquid medium containing
the microorganisms, a carbonator connected to the inlet of the solar
sensor for supplying the liquid medium with the CO.sub.2 necessary for the
photosynthesis, as well as a degasifier connected to the photobioreactor
for eliminating from the liquid medium the oxygen produced by the
microorganisms, as well as the CO.sub.2 not dissolved in the liquid
medium.
The carbonator makes it possible to ensure an adequate transfer of CO.sub.2
from the gas phase to the liquid phase, so that the biological demand for
CO.sub.2 is always satisfied. CO.sub.2 is not then a limiting factor in
the production of the microorganisms.
If the other culture conditions are favorable, such as light, the nutrient
medium, the pH, etc., there is a high demand for CO.sub.2, so that a large
amount of oxygen is formed. In addition, in the solar receiver of the
photobioreactor, there is an enrichment of the culture with dissolved
O.sub.2, as well as gaseous O.sub.2 pockets.
FR-A-2 621 323 teaches elimination by means of a degasifier of the oxygen
produced by photosynthesis prejudicial to the operation of the
photobioreactor and in particular the good heat regulation thereof (drying
of the microorganism and poor photobioreactor efficiency). In addition,
oxygen can be toxic for certain microorganisms.
Contrary to the teaching of the aforementioned document, the inventors have
found that the collection of the oxygen produced by photosynthesis and its
reinjection in the culture medium makes it possible to obtain a
significant antioxidant production by the cultured microorganism. Thus,
under high oxygen content conditions, the photosynthetic microorganisms
react to the oxydizing conditions by synthesizing antioxidants.
The invention therefore relates to a process for the production and
extraction of antioxidants, comprising the following stages:
(a) culturing in a closed photobioreactor of photosynthetic microorganisms
suspended in a liquid culture medium, the oxygen produced by the
microorganisms by photosynthesis being collected and reinjected into the
culture medium, said microorganisms being chosen from the group
constituted by microalgae and cyanobacteria,
(b) separation of the microorganisms from the culture medium,
(c) dissolving the microorganisms separated in (b),
(d) "crushing" the dissolved microorganisms,
(e) addition of solvent to the solution obtained in (d) in order to
solubilize the antioxidants produced by the microorganisms and then
(f) separation of the liquid and solid phases present.
Advantageously, the oxygen is reinjected under pressure into the liquid
medium.
the antioxidants produced are of different types and are a function of the
cultured species.
The microorganisms to which the invention apply are all microalgae and
cyanobacteria which are known. The microalgae are in particular
rhodophyceae such as Porphyridium cruentum which, under oxidizing
conditions, essentially permits Mn SOD production. This SOD is constituted
by two subunits each of 20,000 molecular weights, which are linked by
non-covalent bonds. It has a mean isoelectric point of 4.2.
On average, Porphyridium cruentum produces 10 to 20 U, according to
analysis of NBT (nitroblue tetrazolium), per milliliter of culture (1 ml
of culture containing approximately 1 mg of dry matter of microalgae).
Porphyridium cruentum also makes it possible to produce vitamin C. Thus,
this microalga contains 5 to 7 g/kg of vitamin C.
In this microalga, the "delta-amino-levulinic" metabolic route is very
highly developed and leads to specific pigments (phycobiliproteins) and
two SOD.
However, the culture of microalgae of the chlorophyceae type such as
Haematococcus pluvialis essentially permits in the sugar oxidizing medium
the production of tocopherals and in particular alpha-tocopherals (vitamin
E) at a concentration of 4 to 5 g/kg of microalgae and gamma-tocopherals
at a concentration of 0.5 to 1 k/kg of microalgae. In this microalga, the
"mevalonic" metabolic route is very highly developed.
The conditions favorable for the production of antioxidants according to
the invention are high oxygen production conditions, i.e. high
photosynthetic activity. In addition, advantageously, the culture of
microorganisms is carried out under conditions of natural lighting and the
extraction of the antioxidants from the culture medium, and consequently
the stage (b) of separating the microorganisms from the liquid medium are
carried out in the afternoon.
In addition, the most favorable conditions for the production of
antioxidants are those where the microorganisms are in the exponential
growth phase, i.e. when the growth rate is at a maximum.
For continuous or batch operation, the exponential phase is at the start of
culturing. The microorganisms are in the active division phase, which
corresponds to a maximum photosynthetic activity. In the case of
continuous culturing, it is necessary to choose a rapid replacement rate
for the culture medium (0.2 to 1 every day) in order to maintain the
microorganisms in the active division phase.
Before separating the microorganisms from the culture medium, it is
possible to carry out one or more treatments of the suspension of the
microorganisms with a view to increasing the culture medium content of
antioxidants and in particular SOD by increasing the membrane permeability
of the cells.
This or these treatments can consist of heating an cellular suspension for
a few minutes at between 40.degree. and 50.degree. C. or an ionization of
said suspension with gamma rays. A treatment of 10 kgrays can lead to a 20
to 100% antioxidant activity enrichment of the medium.
Stage (b) of separating the microorganisms from the culture medium serves
to separate the biomass, the intracellular antioxidants being extracted
during stages (c) and (f) from the culture medium, which can contain
exocellular antioxidants.
In the particular case of SOD's, more than half the enzymes are in the
culture medium and the activity measured therein can, in certain cases,
justify the treatment of the liquid medium in order to extract thereform
the exocellular antioxidants.
The dissolving of the microorganisms (stage c) makes it possible to adjust
the concentration of the juice of the microorganisms with a view to
improving the crushing conditions. The optimum microorganism concentration
for the crushing stage is between 20 and 100 g/liter of dry matter, as a
function of the species used and their growth stage.
The "crushing" stage serves to shatter or burst the microorganisms in order
to render the entire cellular content accessible to the solvent. It is
carried out in a homogenizer, where the microorganisms are subject to an
alternation of pressures and vacuums.
The solvent or solvent mixtures used in stage (e) is dependent on the
antioxidants which it is wished to extract. In particular, the SOD's and
vitamin C are hydrosoluble and their extraction takes place in aqueous
solution, whereas the tocopherals are liposoluble and their extraction
takes place in an organic solvent of the acetone or methanol type or in a
vegetable or mineral oil.
If it is wished to extract both liposoluble and hydrosoluble antioxidants
produced by the same species, an organic solvent or an oil is used.
The phase separation stage (f) serves to separate the mixture into two or
three phases as a function of whether or not an aqueous solvent is used:
an aqueous solution containing hydrosoluble antioxidants a lipid solution
containing liposoluble antioxidants and a solid phase constituted by
cellular residues. This phase separation can be carried out by
centrifuging or by decanting in a pulsed column.
Preferably, the crushing, addition of solvent and separation of phases
stages are carried out at approximately 4.degree. C. If necessary, the
lipid and aqueous phases obtained during stage (f), as well as the liquid
medium resulting from the separation stage (b) for the microorganisms can
be concentrated and optionally purified. Said concentrations can be
carried out by ultrafiltration and/or by precipitation with ammonium
sulphate. Purification is advantageously carried out at 4.degree. C. and
its degree is dependent on the use of antioxidants.
The invention also relates to a closed photobioreactor for the production
of antioxidants from a culture of photosynthetic microorganism suspended
in a liquid medium, said microorganisms being chosen from the group
constituted by microalgae and cyanobacteria, said photobioreactor
comprising:
(A)--A solar receiver transparent to sunlight in which circulates the
liquid medium,
(B)--a column carbonator connected to the intake of the solar receiver for
supplying the liquid medium with the CO.sub.2 necessary for
photosynthesis,
(C)--a collector of the oxygen produced by the microorganisms by
photosynthesis and
(D)--means for reinjecting the collected oxygen with respect to the
carbonator.
To obtain a maximum photosynthetic activity, the solar receiver comprises
parallel tubes in which circulates the liquid medium, end collectors
ensuring the linking of the said tubes.
BRIEF DESCRIPTION OF DRAWINGS
The invention is described in greater detail hereinafter relative to
non-limitative embodiments and the attached drawings, wherein show:
FIG. 1 diagrammatically the different stages of the extraction process
according to the invention of antioxidants from a culture of microalgae.
FIG. 2 diagrammatically a photobioreactor for the production of microalgae
according to the invention and operating in batch manner.
FIG. 3 the evolution of the photosynthetic activity in the culture medium
during one day.
FIG. 4 diagrammatically a photobioreactor for the production of microalgae
according to the invention and operating continuously.
As shown in FIG. 1, the Porphyridium cruentum culture 1 takes place in a
photobioreactor 2 containing as the nutrient medium synthetic sea water
enriched with potassium, phosphorus and nitrogen. The potassium is present
in chloride form, the phosphorus in orthophosphoric acid form and the
nitrogen in the form of urea and nitrates.
The pH of the culture medium is regulated between 6 and 8 throughout the
growth of the microalgae and the carbon supply takes place in the form of
carbon dioxide. The precise construction of this photobioreactor will be
described relative to FIGS. 2 and 3.
The culture of microalgae takes place over several days and the culture
medium contains approximately 1 g/liter of dry matter of microalgae. The
production of these microalgae takes place under natural lighting
conditions and the treatment of the microalgae for extracting therefrom
the antioxidants lasts between 12 and 18 hours and in an exponential
growth phase for the same.
The culture removed from the photobioreactor is placed in a water bath 4
heated to between 40.degree. and 50.degree. C. for 10 to 20 minutes, with
a view to increasing the membrane permeability of the microalgae and
consequently the content of exocellular antioxidants. This heating is
accompanied by stirring.
As indicated at 6, the culture 1 is then filtered on a rotary filter 7 with
a flow rate of close to 200 l/h/m.sup.2, in order to separate the
microalgae from the liquid medium. This stage can also be performed on a
continuous centrifuge between 10,000 and 30,000 g with a flow rate of 1
m.sup.3 /h. The clear filtrate 9 obtained in 6 contains more than half
superoxide-dismutase enzymes. In particular, the enzymatic activity
measured in the liquid medium is 10 to 30 U/m, the measurement being
carried out by NBT.
The SOD's are directly in aqueous solution can then be concentrated in the
manner indicated at 8 by ultrafiltration with a 10,000 daltons mineral
membrane. The thus concentrated filtrate 9 can be purified, as indicated
at 10 and in particular by anion exchange chromatography. The stages of
concentration 8 and purification 10 are performed at approximately
4.degree. C.
The solid product obtained 6 essentially contains microalgae, which are
diluted, accompanied by stirring and as indicated at 12, in a phosphate
buffer solution (50 mM, pH 7.8) introduced at 13, in order to obtain an
optimum microalgae concentration for the "crushing" stage 14. The
microalgae concentration in the suspension 15 obtained at 12 is 20 to 100
g/l of dry matter (dm).
The suspension 15 is then subject to a pressure-vacuum cycle in an ATV
homogenizer of the Manton-Gaulin type operating at a pressure of 2 to
5.10.sup.7 Pa. This stage is preferably carried out at 4.degree. C. It is
used for shattering the microalgae, so that the entire cellular content is
accessible to the solvents.
To the cellular suspension of Porphyridium cruentum obtained in 14 is added
a vegetable or mineral oil, e.g. a soy oil or a solvent of the acetone or
methanol type in order to extract both the liposoluble antioxidants such
as the tocopherols and the hydrosoluble antioxidants such as vitamin C and
SOD.
This solvent addition is represented at 16 and the arrow 17 indicates the
solvent supply to the cellular suspension. This solvent addition takes
place accompanied by stirring and at approximately 4.degree. C. The
cellular suspension obtained carries the reference 19.
As indicated at 18, the cellular suspension 19 is then centrifuged in a
single-stage centrifugal extractor equipped with type VA 35-09-566 piston
valves. The centrifugate obtained is particularly constituted by an
aqueous solution containing SOD and vitamin C and a lipid solution
containing tocopherols. Reference 21 corresponds to the recovery of the
aqueous solution and reference 23 to the recovery of the lipid solution.
These solutions can then be concentrated as indicated at 8, in the same
way as the liquid medium obtained after separating the microalgae in 6.
The solid residues obtained at 25 and resulting from centrifuging 18
contain fragments of microalgae.
FIG. 2 shows a photobioreactor according to the invention used for the
batch culturing of microalgae or cyanobacteria, which produce
antioxidants. The closed photobioreactor 2 has a solar receiver 30,
constituted by parallel, flexible, transparent plastics material tubes 32,
such as of polyethylene, in which circulates the culture medium 1
containing the microorganisms and the nutrient elements necessary for the
growth thereof. The polypropylene collectors 34 and 36 make it possible to
interconnects the tubes 32 and ensure the passage of liquid from one tube
to the other. This solar receiver 30 is intended to be placed on the
surface of an expanse of water for its heat regulation.
For further details concerning the construction and operation of the solar
receiver or sensor reference can be made to the aforementioned FR-A-2 621
323.
The CO.sub.2 supply for the culture medium during a cycle or run is
provided by a carbonator 38 connected to the intake of the solar sensor
30, via a supply pipe 40. The latter is equipped with a pump 42 ensuring
the circulation of the culture medium throughout the photobioreactor. The
carbonator 38 is of the column type and the CO.sub.2 supply for the
culture medium via the pipe 40 takes place through the bottom of the
carbonator.
The photobioreactor also has a vessel 44 used for the preparation,
accompanied by stirring, of the nutrient medium necessary for the growth
of the microalgae. This nutrient medium is introduced at the start of the
run into the liquid medium containing the microalgae, using a supply pipe
46 branched to the supply tube 40. The branch pipe 46 is equipped with a
valve 47 making it possible to control or check the injection of the
nutrient medium.
In the solar receiver 30, the photosynthesis reaction leads to the
formation of oxygen. Purge tubes 48 fitted at the outlet of the solar
receiver 30 and more specifically at the outlet of the collectors 34 and
36 on make it possible to eliminate the gas pockets which form in the
solar receiver tubes 32. This gas can contain up to 75% by volume of
oxygen under high sunlight conditions for the solar receiver 30, said
oxygen resulting from the photosynthesis.
This gaseous oxygen is collected in a collector 50 and then reinjected into
the carbonator 38 with the aid of a pipe 49. The recycling of the gaseous
oxygen takes place at the bottom of the carbonator 38.
In said carbonator, the oxygen is redissolved in the culture medium. In
order to assist the passage of the oxygen into the culture medium, the
upper part of the carbonator is equipped with a conical deflecting plate
51.
The supply of CO.sub.2 into the carbonator 38 is carried out by mixing with
air up to 80% by volume of CO.sub.2. This gaseous mixture is introduced
into a vessel 52 via a supply pipe 53.
The outlet of the said vessel is equipped with a supply pipe 54 connected
to a central immersion tube 56 of the carbonator 38 and positioned along
the axis of revolution of the latter. The immersion end 58 of the tube 56
is made from fritted or sintered stainless material or glass having a
variable porosity in order to improve the countercurrent dissolving of the
CO.sub.2 in the liquid medium containing the microalgae. The introduction
of the culture medium 1 into the carbonator takes place in the upper part
of the latter by means of a supply pipe 60 connected to the outlet of the
downstream collector 36 of the solar receiver.
The air-CO.sub.2 mixture introduced into the carbonator 38 through the
fritted glass 58 causes a slight desorption of the oxygen dissolved in the
culture medium. The gaseous mixture discharged at the top of the
carbonator is recovered by a discharge pipe 62 for reinjection into the
vessel 52.
The gas from the said vessel 52 is compressed by a piston-type device 64
and reinjected into the carbonation column 39 via the pipe 54 and then the
immersion tube 56. The said gas reinjected at the bottom of the carbonator
contains desorbed oxygen and CO.sub.2 not dissolved in the liquid phase.
The vessel 52 is equipped with a pressostat 66, whose pressure level is
regulated between 0.1 and 0.5.10.sup.5 Pa. This system leads to a
dissolved oxygen enrichment of the culture.
Under high sunlight conditions, the oxygen percentage in the gaseous phase
is 21% by volume and at the intake of the supply pipe 53.
At the outlet from the carbonator 58 or at the inlet of the solar receiver
30, i.e. in tube 40, the gaseous mixture contains 70% by volume oxygen.
This is due to the fact that the culture arriving at 60 in the carbonator
is very rich in dissolved oxygen resulting from the photosynthesis. At the
outlet from the solar receiver, i.e. in the purge tubes 48, the gaseous
mixture contains 75% by volume of oxygen. Thus, there is a 70 to 75% by
volume oxygen enrichment in the solar receiver 30 as a result of the
confinement of the culture medium.
The partial deoxygenation or oxygen desorption lowers the oxygen content of
the gaseous phase by 70 to 75%. Under these oxidizing conditions, the
cultured microalgae react by synthesizing antioxidants.
In particular, the microalga Porphyridium cruentum reacts by producing SOD
at a mean rate of 10 to 20 U (NBT)/ml of culture, i.e. 5.times.10.sup.6 to
20.times.10.sup.6 U/m.sup.3 and vitamin C at a rate of 5 to 7 g/kg.
However, the microalga Haematococcus pluvialis reacts by producing vitamin
E at a rate of 4 to 5 g/kg of microalgae and gamma-tocopherol at a rate of
0.5 to 1 g/kg of microalgae.
The apparatus shown in FIG. 2 also has a branch pipe 68 equipped with a
valve 70 mounted on the outlet pipe 60. The pipe 68 issues into a vessel
70 used for collecting the microalgae and the culture medium after a
culture cycle with a view to extracting the antioxidants therefrom.
The conditions favorable for the production of antioxidants are high
photosynthetic activity conditions. It is also possible to optimize these
conditions on a scale of a 10 to 20 days culture cycle or run. The
photosynthetic activity of the microalgae is at a maximum at the start of
the run, i.e. in the exponential growth phase and this is illustrated by
the following example.
During a run carried out on a Porphiridium cruentum culture, the inventors
noted the following SOD contents:
5th day:
25 U (NBT)/ml of culture
(1.0 g dm/l of culture), i.e.
25,000 U/g of culture.
10th day:
31 U (NBT)/ml of culture
(1.3 g dm/l of culture) i.e.
24,000 U/g of culture.
17th day:
27 U (NBT)/ml of culture
(1.8 g dm/l of culture) i.e.
15,000 U/g of culture.
26th day:
3 U (NBT)/ml of culture
2.0 g dm/l of culture) i.e.
1,500 U/g of culture.
This example makes it clear that the maximum antioxidant production takes
place between the 1st and 10th days of a run.
Similar measurements were carried out during a run with Heamatococcus
pluvialis as the microalga. The inventors noted that following tocopherol
contents in the microalgae:
2nd day:
4,500 ppm of alpha-tocopherol
660 ppm of gamma-tocopherol
20th day:
80 ppm of alpha-tocopherol
130 ppm of gamma-tocopherol.
According to the invention, the microorganisms are cultured in the closed
photobioreactor under natural lighting conditions, i.e. with an
alternation of daylight and night. The assimilation of CO.sub.2 by these
microorganisms and consequently the production of O.sub.2 are directly
linked with the light intensity. Therefore they are at an optimum at
midday.
The graph of FIG. 3 reveals the evolution of the dissolved CO.sub.2
pressure and the dissolved oxygen pressure in the culture medium during
one day. The pressures of these gases are given in parts per million.
Curve A gives the CO.sub.2 pressures and curve B and O.sub.2 pressures.
Curve C gives the solar energy ES during the day and is expressed in
kilojoules/m.sup.2 /s.
These curves make it clear that the best antioxidant production conditions
occur between midday and 6 p.m., so that the antioxidants are collected
during this period.
The culture of the microalgae and therefore the production of antioxidants
can also take place continuously with the aid of the photobioreactor shown
in FIG. 4. The constituent elements of the said photobioreactor identical
to those of the photobioreactor of FIG. 2 carry the same references.
As in FIG. 2, the photobioreactor has a tubular solar receiver 30 equipped
with upstream and downstream collectors 34 and 36. A pump 42a mounted on a
pipe 40a links the inlet and outlet of the collector 34 and functions in a
continuous manner. It ensures the continuous circulation of the culture
medium containing the microalgae in the solar receiver 30. The assembly
constituted by the solar receiver, the pipe 40a and the pump 42a forms a
closed or sealed assembly, i.e. the microorganisms do not pass out of it.
The nutrient medium prepared accompanied by stirring in the vessel 44 is
introduced continuously in the present case at the top of the carbonator
38a by means of a supply pipe 46a equipped with a continuously operating
pump 73, which also regulates the flow rate of the nutrient medium and
therefore the nutrient medium replacement rate (once daily).
The immersion tube 56, equipped with its fritted glass 58 at its immersed
end, makes it possible to supply CO.sub.2 in countercurrent with the
nutrient medium to the carbonator 38a.
In order to improve the dissolving of the CO.sub.2 gas injected under
pressure by means of the tank 74 fitted at the inlet of the immersion tube
56, the column of the carbonator is equipped with a lining 76 of the
Raschig ring or Bert support type.
The carbonated nutrient medium is then injected into the solar receiver 30
by a supply pipe 40b linking the base of the carbonator 30a to the
collector 34 of the solar receiver 30. The said pipe 40b is equipped with
a continuously operating pump 42b regulated to the same flow rate or
delivery as the pump 73 and 75.
At the outlet from the solar receiver 30, a pipe 60a equipped with pump 75
makes it possible to continuously supply of the culture medium containing
the microorganism to a collecting vessel 77, with a view to the extraction
of the antioxidants.
As previously, in the solar receiver 30, the photosynthesis brings about a
dissolved oxygen enrichment of the culture. The oxygen-containing gas
pockets formed are discharged from the receiver 30 by purge tubes 48 in
the direction of the oxygen collector 50, which is equipped with a
manometer 78 for measuring the pressure of the oxygen-enriched purge gas.
When the pressure is between 0.4 and 0.5.10.sup.5 Pa, an electrovalve 80
releases the accumulated gas into the collector 50. This gas is then
reinjected by the pipe 49 at the bottom of the carbonator 38a, in such a
way that the gaseous oxygen resulting from the photosynthesis dissolves in
the culture medium. As previously, the accumulation of the gas in the
solar receiver 30 causes a dissolved oxygen enrichment.
In order to increase the dissolved O.sub.2 concentration in the liquid
medium of the photobioreactor, it is desirable to make it operate under an
overpressure. In the example shown, the solar receiver tubes 32 are unable
to resist a pressure above 0.5.10.sup.5 Pa. In addition, the triggering of
the electrovalve 80 takes place at between 0.4 and 0.5.10.sup.5 Pa. The
normal pressure of the oxygen-enriched gas is between 0.1 and 0.2.10.sup.5
Pa.
In the photobioreactor shown in FIG. 2, the carbonator 38 does not have a
lining, unlike in the case of the carbonator 38a of FIG. 4 due to the fact
that in batch operation, the liquid medium containing the microorganisms
passes through the carbonator and would adhere to the lining. Only the
gases and nutrient medium circulate in the carbonator 38a.
In order to further improve the oxygen enrichment of the culture medium, it
is possible to replace the pressurized CO.sub.2 tank 74 of the
photobioreactor of FIG. 4 by the recovery system 52-54-66 for the oxygen
desorbed in the carbonator shown in FIG. 2.
With Porphyridium cruentum cultured and treated according to the invention,
it is possible to obtain 5.times.10.sup.6 to 20.times.10.sup.6 NBT units
of SOD in 1000 liters of culture, which corresponds to the production of 9
to 36 g of SOD/m.sup.3 of culture. Under these conditions, solar receivers
30 distributed over a one hectare water surface and operating for 200 days
every year will make it possible to produce 90 to 360 kg or SOD.
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